This application consists of two major sections. In the first, we propose experiments directed at understanding the control of cell-type gene expression and differentiation of prestalk and prespore cells. Cis-acting regulating regions will be identified using DNA-mediated transformation combined with in vitro mutagenesis approaches. Sequence-specific DNA binding proteins will be examined with the goal of understanding if common regulating proteins are involved in controlling these gene sets. Potential regulating mutants will also be isolated. Using immunostaining and in situ hybridization we will examine where within the developing aggregate prestalk and prespore genes are first expressed to understand how the spatial patterning of cells is established. The regulation and function of the Dictyostelium ras gene, a prestalk-specific gene, will be examined during development. The cis-acting regulating regions of this gene will be defined and possible functions of the protein in prestalk differentiation will be examined. In the second project, we examine the control of the 17-20 member, developmentally regulated, multigene family encoding actin. The relative expression of the 15 cloned actin genes will be examined. Cis-acting control regions of a few selected actin genes will be identified. The function of identified, conserved GC-rich sequences within the 5' flanking region will be examined to determine if they are important in regulating the expression of specific actin genes. DNA binding proteins specific for identified regulating sequences will be isolated and binding sites mapped. These experiments are aimed at understanding the complex regulating pattern of this essential gene family.